Primary lymphedema is the cl inical expression of defects in lymphatic development and function. Three genes have been shown to cause primary lymphedema, VEGFR3, FOXC2 and SOX18. Mutations in these genes account for only a fraction (-1 /3) of all cases of primary lymphedema. Recently, we found strong evidence that mutations in hepatocyte growth factor (HGF) and its high affinity receptor MET cause primary lymphedema in patients and families. The goals of this grant are to characterize these mutations molecularly and functionally in lymphatic endothelial cells in culture, and to use the zebrafish as a model for the in vivo ascertainment of the effects of these mutations on lymphatic development. This goal will be met by fulfilling the following specific aims 1) To assay the functional consequences of the lymphedema associated HGF/MET mutations in 2-D and 3-D lymphatic endothelial cell culture with regard to proliferation, mobility, gap junction communication, cell survival and tubulogenesis. 2) To assay the HGF/MET mutations by assessment of MET activation and phosphorylation status. 3) To determ ine the consequences of these prototypical mutations in HGF/MET on vertebrate Iymphangiogenesis by analyzing a) their function by RNA expression and antisense morpholino analysis in fluorescent (fl i-GFP) zebrafish embryos;and b) th e effect of small molecule inhibitors of MET on lymphatic development. 4) To recruit the extended families of those pro bands with HGF and MET mutations to carry out genotype X phenotype corre lations in families who carry these mutations. These studies will reveal th e function of the HGF/MET system in lymphatic biology, in lymphedema and particularly inform strategies to prevent and treat primary and secondary lymphedema.